Induction of IGHV3-53 public antibodies with broadly neutralising activity against SARS-CoV-2 including Omicron subvariants in a Delta breakthrough infection case

Abstract

Background: Emergence of SARS-CoV-2 variants that escape neutralising antibodies hampers the development of vaccines and therapeutic antibodies against SARS-CoV-2. IGHV3-53/3-66-derived public antibodies, which are generally specific to the prototype virus and are frequently induced in infected or vaccinated individuals, show minimal affinity maturation and high potency against prototype SARS-CoV-2.

Methods: Monoclonal antibodies isolated from a Delta breakthrough infection case were analysed for cross-neutralising activities against SARS-CoV-2 variants. The broadly neutralising antibody K4-66 was further analysed in a hamster model, and the effect of somatic hypermutations was assessed using the inferred germline precursor.

Findings: Antibodies derived from IGHV3-53/3-66 showed broader neutralising activity than antibodies derived from IGHV1-69 and other IGHV genes. IGHV3-53/3-66 antibodies neutralised the Delta variant better than the IGHV1-69 antibodies, suggesting that the IGHV3-53/3-66 antibodies were further maturated by Delta breakthrough infection. One IGHV3-53/3-66 antibody, K4-66, neutralised all Omicron subvariants tested, including EG.5.1, BA.2.86, and JN.1, and decreased the viral load in the lungs of hamsters infected with Omicron subvariant XBB.1.5. The importance of somatic hypermutations was demonstrated by the loss of neutralising activity of the inferred germline precursor of K4-66 against Beta and Omicron variants.

Interpretation: Broadly neutralising IGHV3-53/3-66 antibodies have potential as a target for the development of effective vaccines and therapeutic antibodies against newly emerging SARS-CoV-2 variants.

Funding: This work was supported by grants from AMED (JP23ym0126048, JP22ym0126048, JP21ym0126048, JP23wm0125002, JP233fa627001, JP223fa627009, JP24jf0126002, and JP22fk0108572), and the JSPS (JP21H02970, JK23K20041, and JPJSCCA20240006).

Keywords: Neutralising antibody; Public antibody; SARS-CoV-2; Variant.

Fig. 1

Isolation of mAbs from the patient with Delta BTI, KUKMC-19–22. (a) Summary of patient information for KUKMC-19–22, who donated blood samples for isolation of mAbs by single-cell sorting and construction of recombinant IgGs. (b) Neutralising activity of plasma from KUKMC-19–22 at 18 days post symptom onset, analysed using pseudoviruses carrying S proteins from variants 614G, Beta, and Delta. (c) Single-cell sorting of IgG⁺IgM⁻ B cells, with or without RBD probes from the Wuhan strain and Beta variant. (d) Summary of mAbs isolated from KUKMC-19–22. The supernatants from the transfected cells were analysed for reactivity to the Wuhan strain S protein using flow cytometry and neutralising activity against the 614G variant.

Fig. 2

Characterisation of mAbs isolated from the Delta BTI case. (a) Characteristics of neutralising mAbs, of which 23 mAbs were purified by Protein A and examined for neutralising activities against pseudoviruses carrying S proteins from the 614G, Beta, and Delta variants and Omicron subvariants BA.1, BA.2, BA.4/5, and XBB.1.5. Sorting was performed with RBD probes (RBD+) or CD27 (CD27+). Lineages of mAbs are summarized in Supplementary Table S2. DPSO: Days post symptom onset; SHM: Somatic hypermutation %; ND: not done. (b) Plot of IC₅₀ values of 23 mAbs against SARS-CoV-2 variants. P values calculated using the Mann–Whitney test; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (c) IC₅₀ values of mAbs using IGHV3-53/3-66 (n = 6, left) and IGHV1-69 (n = 6, right) against the SARS-CoV-2 614G and Delta variants. P values calculated using the Mann–Whitney test; ∗P < 0.05, and ns: not significant. (d) Rates of SHM (%) of mAbs using IGHV3-53/3-66 (n = 7, blue), IGHV1-69 (n = 6, red), or other genes (n = 12, green).

Fig. 3

Neutralisation breadth of the K4-66 lineage antibodies. (a) Amino acid sequences of K4-66 lineage antibodies K1-68 and K4-66 aligned with those of their germline genes, IGHV3-53∗04 and IGKV1-33∗01. (b) IC₅₀ values of K4-66, K1-68, and K4-66IG, the inferred germline precursor of K4-66 against SARS-CoV-2 variants. (c) Neutralising activities of K4-66 mutants against pseudoviruses carrying S proteins from the 614G, Beta, and Delta variants and Omicron subvariants BA.1, BA.2, BA.4/5, XBB.1.5, and XBB.1.16. IC₅₀ values (μg/mL) and the fold change (IC₅₀ value of 4-66IG/IC₅₀ value of mutant) are shown in the upper and lower rows, respectively. K4-66IGK (H-chain from WT K4-66 and L-chain from the inferred germline-reverted K4-66 [H: WT, L: IG]), K4-66IGH (H-chain from the inferred germline-reverted K4-66 and L-chain from WT K4-66 [H: IG, L: WT]), and K4-66IG mutants containing F28I, F28L, A96G, and A96V in the H-chain were compared with K4-66IG and K4-66. NA; not available. (d) NT₅₀ values of plasma samples from patient KUKMC-19–22 and seven other patients with Delta BTI (see Supplementary Table S1) against SARS-CoV-2 variants.

Fig. 4

Neutralising activity of mAbs against various SARS-CoV-2 variants. IC₅₀ values (μg/ml) of mAbs against pseudoviruses carrying S from 614G, Beta, and Delta variants, and various Omicron subvariants. In addition to five mAbs from patient KUKMC-19–22, two mAbs isolated from two convalescent patients infected with prototype SARS-CoV-2 (previous study), and five therapeutic mAbs were examined for neutralising activities.

Fig. 5

Binding affinity of mAbs against the Wuhan strain and Omicron subvariant XBB RBDs. (a) Representative SPR sensorgrams of K4-66 binding affinity to Wuhan and XBB RBDs. Mean k_a and k_d values from three replicates are also shown. (b) Mean K_D values of binding affinity of four mAbs to Wuhan and XBB RBDs. Binding affinity was determined by SPR analysis using Biacore T200.

Fig. 6

Neutralising activity of mAbs against authentic SARS-CoV-2 variants. (a) IC₅₀ values of mAb neutralisation of authentic variants as determined by plaque assay. (b) Representative results of neutralisation by K4-66. Serially diluted K4–66 was mixed with the Omicron subvariant BA.5 (TKYTS14631), and added to VeroE6/TMPRSS2 cells for plaque formation.

Fig. 7

Effect of mAb K4-66 on infection with Omicron subvariant XBB.1.5 in hamsters. Syrian hamsters were inoculated with 10³ PFU of XBB.1.5 and intraperitoneally administered with 5 mg/kg K4-66 (n = 5) or normal human IgG (n = 5, control) 1 day later. Viral load titre was determined by PFU (a) and quantitative PCR (b) at 4 days post infection in the lungs (left) and nasal turbinates (right). (c) Serum neutralisation titres against Wuhan (left) and XBB.1.5 (right) in plasma samples at 4 days post infection. (d) Representative images of the lungs of infected hamsters stained by haematoxylin and eosin (H&E). (e) The extent of inflammatory cell infiltration in the lungs. (f) Representative images of the lungs stained by anti-SARS-CoV-2 nucleocapsid protein. (g) The extent of viral antigen-positive cells in the lung. The scores were determined as follows: 0, no change; 1, mild; 2, slight; 3, moderate; 4, severe. P value was calculated using the Mann–Whitney test. ∗P < 0.05, ∗∗P < 0.01, and ns: not significant.

Fig. 8

Structural analysis of SARS-CoV-2 XBB.1.5 S and Fab K4-66 complex. (a) Overall cryo-EM map of XBB.1.5 S trimer in complex with Fab K4-66. Each protomer in the S trimer is colored by green, red, and blue, while the Fab is dark blue. (b) Cryo-EM map of the locally refined RBD-Fab complex with the rigidly fitted atomic models of XBB.1.5 S RBD (orange, PDB ID: 8JYP) and Fab K4-66 (PDB ID: 9II9). Structures are shown in ribbon representation. H- and L-chains are shown in sky blue and light blue, respectively. XBB.1.5 RBD is shown in orange. (c) Crystal structure of Fab K4-66. Same colors as in Fig. b. (d) Superimposed structures of Fab K4-66-RBD complex with Fab CC12.1-RBD complex (grey, PDB ID: 6XC2). (e) Electrostatic surface of the paratope and epitope. Surface is visualized using APBS plugin in PyMOL. (f) Structure of Fab K4-66 and close-up views of the residues substituted from the inferred germline sequence that are important for neutralising activity (Fig. 3).